The introduction of whole-genome sequencing has provided researchers with an unprecedented ability to measure the complex state of genomic rearrangements characteristic of most cancers. Numerous methods for inferring structural variation from paired-end sequencing data have been developed (Bioinformatics (2009); 25: i222-i230; Nature Methods 2009 August; 6:677-681; Nature Genetics 2011 March; 43:964-968), but the structural variants called by such methods are often considered only in isolation, used primarily to identify potential fusion genes. The difficulty in discovering all true structural variants and filtering out false positives makes it hard to use the output of currently known methods to reassemble large regions of the tumor genome. Such difficulties are particularly unfortunate as proper tumor genome assemblies help reveal the complex structure of the tumor genome and could be used to infer a mechanism by which somatic alterations such as amplifications of oncogenes and deletions of tumor suppressors occur.
Rapidly decreasing cost and increased data resolution of whole-genome sequencing also promises the emergence of new classes of cancer diagnostics from blood. For example, Leary et al. (SciTransl Med 2010 February; 2(20): 20ra14) developed a personalized analysis of rearranged ends (PARE), which uses somatic rearrangements to build a blood-based diagnostic assay for recurrence. While this novel method provides a powerful framework for monitoring, analysis of biopsied tumor tissue is typically needed to find specific markers to be measured in blood. Other monitoring techniques, such as measuring circulating tumor cells, require significant enrichment efforts that are only feasible when tumors with metastatic potential are present (Cancer Lett. 2007 August; 253(2): 180-204). Both of these techniques present technical challenges that make them unsuitable for initial tumor diagnosis.
It is well documented that double-stranded DNA can become highly amplified and circularized in the cytoplasm of cells, forming what is known as double minutes (Cancer Genet. Cytogenet. 1982 February; 5(1): 81-94). Double minutes (DMs) have been shown to confer resistance to certain drugs, as well as pass along this resistance non-uniformly to daughter cells. They have been observed up to a few megabases in size, and contain chromatin similar to actual chromosomes, but lack the centromere or telomeres found in normal chromosomes. Since DMs lack centromeres, they are randomly distributed to daughter cells during cell division, and they are generally lost in future generations unless there is some selective pressure to maintain them. However, the random distribution of DMs also provides a simple mechanism to quickly amplify an oncogenic DM in successive generations, where cells may accumulate hundreds of copies of the double minute. Though the frequency of double minutes in glioblastoma multiforme (GBM) is largely unknown, a recent study by Fan et al. (J. Appl. Genet. 2011 February; 52(1): 53-59) has identified neuroblastomas as having the second highest rate of DMs, offering the possibility that perhaps some of the frequently amplified oncogenes in GBM tumors can be explained by the formation and accumulation of oncogenic double minutes.
Despite the fact that DMs were originally identified over thirty years ago, there is no evidence in the literature that a comprehensive sequence analysis of DMs has been done. Thus, there is still a need for improved diagnostic methods, and especially improved methods for genetic analysis of neoplastic tissue that may be associated with presence of DMs.